Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human α-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including α-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson9s disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30–80% of the nigral dopamine neurons, a 40–50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50–60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that α-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant α-synuclein, pointing to a key role for α-synuclein in the pathogenesis of Parkinson9s disease. Targeted overexpression of α-synuclein in the nigrostriatal system may provide a new animal model of Parkinson9s disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

https://www.jneurosci.org/content/jneuro/22/7/2780.full.pdf

Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system.

Since the finding by [Johnson and Ascher (1987)][1] demonstrating that glycine enhances electrophysiological responses mediated by N-methyl-d-aspartate (NMDA)b-sensitive glutamatergic receptors, considerable interest has been devoted to this topic (for reviews, see [Dingledine et al. , 1990][2]; [

Glycine and N-Methyl-d-Aspartate Receptors: Physiological Significance and Possible Therapeutic Applications

Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors) and a class of G-protein coupled receptors (metabotropic glutamate receptors). Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases.

/pdf/chronic-glutamate-toxicity-in-neurodegenerative-diseases-m0iiq3qag7.pdf

Chronic Glutamate Toxicity in Neurodegenerative Diseases—What is the Evidence?

Lewy bodies (LB) in the substantia nigra are a cardinal pathological feature of Parkinson's disease, but they occur in a number of neurodegenerative diseases and can be widespread in the nervous system. The characteristics, locations, and composition of LB are reviewed, with particular attention to α-synuclein (α-SYN), which appears to be the major component of LB. The propensity for α-SYN, a presynaptic protein widely expressed in the brain, to aggregate is because of an amyloidogenic central region. The factors that favor the aggregation of α-SYN and mechanisms of toxicity are examined, and a mechanism through which aggregates of α-SYN could induce mitochondrial dysfunction and/or release of proapoptotic molecules is proposed.

https://www.pnas.org/content/pnas/103/6/1661.full.pdf

Lewy bodies

alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.

/pdf/the-parkinson-s-disease-protein-a-synuclein-disrupts-snediinwj7.pdf

The Parkinson's disease protein α-synuclein disrupts cellular Rab homeostasis

We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert-butyl-oxycarbonyl-L-cysteine and o-phthaldialdehyde. Exceptionally high levels of free D-aspartate and D-serine were demonstrated in the fetal cortex at gestational week 14. The ratios of D-aspartate and of D-serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of D-aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of D-serine throughout embryonic and postnatal life, whereas the D-amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because D-aspartate and D-serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these D-amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.

Embryonic Development and Postnatal Changes in Free d‐Aspartate and d‐Serine in the Human Prefrontal Cortex

Neurons containing a central pale area which may possibly represent an early cytoplasmic change before Lewy body maturation were observed in the substantia nigra from a rare autopsy case of juvenile parkinsonism. Ultrastructurally, such neurons exhibited cytoplasmic swelling along with disappearance of the rough endoplasmic reticulum from the central part of the perikaryon. The pale central cytoplasm was replaced by numerous-cored vesicles, mitochondria, ribosomelike granules and a few Lewy body filaments. The relation of this central pale area to the pale body is discussed.

An early cytoplasmic change before Lewy body maturation: an ultrastructural study of the substantia nigra from an autopsy case of juvenile parkinsonism

We evaluated four autopsy cases of dentatorubropallidoluysian atrophy (DRPLA) which had been diagnosed clinically as Huntington's chorea based on choreic movements, mental symptoms, and family history. These cases showed cerebellar ataxia at an early stage and developed choreic movements including a ballistic element. Pathological examination after autopsy revealed findings of DRPLA in all cases, although each case appeared to be in a different stage. Comparison of the clinical signs with the pathological findings suggests that the cerebellar ataxia observed clinically may have been related to pathological changes in the dentate nucleus of the cerebellum, and choreic movements may have been related to the pallidoluysian system, in particular, the nucleus of Luys. Biochemical analysis using frozen brain tissue revealed a decrease in γ-aminobutyric acid (GABA) in the substantia nigra similar to that seen in Huntington's chorea in three cases. The findings of this study help to clarify the pathological processes that are responsible for the motor disturbances seen in patients with DRPLA.

A clinico-pathologico-biochemical study of four autopsy cases of dentatorubropallidoluysian atrophy: With special reference to choreic movement

With the purpose of achieving a better correlation between the neuropathologic features observed by light microscopy (LM) and those seen by electron microscopy (EM), the authors attempted to use a conventional electron microscope (EMe) to obtain images at the extremely low magnification (mag) of 500x. This goal was successfully accomplished by using the methodology devised for this investigation, for which 100 nm thick, 2×2 mm sections were prepared. These were mounted on 75 hexagonal grids (VECO), double-stained for a slightly longer period of time than usual, and then examined with a conventional EMe using the low mag mode lens system and a 100 KV accelerating voltage. The technique was used for the examination of three degenerative structures, to wit: grumose degeneration, stellate body and senile plaques. The present method allowed satisfactory viewing at an extremely low mag, as a given degenerative structure readily fit in its entirety on one sheet of film. The structural features were strikingly more distinct when viewed at low mag EM than at a similar LM mag. For instance, by using the former, the central cores of classical senile plaques had the appearance of chestnut burrs or of a herring-bone pattern with numerous amyloid bundles. Based on their results, the authors believe that the low mag EM technique provides an essential step toward the correlation of LM and high mag EM findings.

Yoshihiko Mizutani

Papers

Embryonic Development and Postnatal Changes in Free d‐Aspartate and d‐Serine in the Human Prefrontal Cortex

An early cytoplasmic change before Lewy body maturation: an ultrastructural study of the substantia nigra from an autopsy case of juvenile parkinsonism

A clinico-pathologico-biochemical study of four autopsy cases of dentatorubropallidoluysian atrophy: With special reference to choreic movement

Extremely Low Magnification Electron Microscopy for Observation of Degenerative Changes of the Central Nervous System

60-year-old female with SLE complicated by progressive ataxia